CAREER: Discovering Upstream Effectors to Cell Fate Determination

在脊椎动物胚胎的正常发育过程中，胚胎的细胞必须发育成有机体的所有不同类型的组织，但有助于组织形成的一整套生物分子尚不清楚。该项目利用最近才在分析化学领域开发的先进仪器来确定小分子的变化，因为在早期发育的青蛙胚胎中，单个细胞形成不同类型的组织，这是脊椎动物胚胎发育的重要模型。由此产生的数据将提供以前无法获得的对细胞、组织、器官和生物体的形成至关重要的基本生物过程的见解。这些研究工作为东道国大学以及会议、研讨会和国家培训中心(如纽约州冷泉港实验室)对不同参与者进行跨学科培训奠定了基础。这项工作将培养生物和化学领域的新一代科学家，包括许多传统上在科学界代表性不足的人群，使他们能够应对当前的挑战，同时也在这些领域提出新的问题，以更好地了解正常的脊椎动物胚胎发育和疾病。大量研究发现了许多在脊椎动物胚胎发育过程中具有关键作用的基因和基因产品，但小分子(称为代谢物)如何参与细胞发育过程尚不完全清楚。PI的实验室最近在早期非洲爪蛙胚胎中发现了能够改变选定干细胞正常背腹命运的代谢物，表明这些分子也是脊椎动物身体模式形成过程中的活跃参与者。这项工作的总体目标是确定代谢物诱导的细胞命运决定的作用机制。这将通过系统细胞生物学方法来实现，在系统细胞生物学方法中，将使用在PI的实验室开发和验证的独特的单细胞质谱学技术来表征代谢物注入细胞的分子状态。该项目将确定改变细胞命运的代谢物如何扰乱近距离代谢网络以及代谢的关键蛋白和已知的背腹规范信号通路。两台单细胞质谱仪将用于对注入的代谢物进行通量分析，并测量目标蛋白在荧光跟踪的细胞克隆中的相对翻译，这些克隆是由活的青蛙胚胎中的代谢物注入的细胞形成的。由此产生的数据将通过基因敲除实验确定功能测试的候选基因，以验证所提出的代谢物诱导细胞命运改变的作用机制。了解小分子对细胞命运承诺的影响在生命科学的不同领域产生了广泛的影响。这项工作还将培训未被充分代表的群体掌握连接生物学和分析化学的技术。

Non-Technical ParagraphDuring normal development of the vertebrate embryo, cells of the embryo must develop into all the different types of tissues of the organism, but the complete set of biomolecules contributing to tissue formation is unknown. This project utilizes advanced instruments that have only recently been developed in analytical chemistry to determine changes in small molecules as individual cells form different types of tissues in the early developing frog embryo, which is an important model of vertebrate embryo development. The resulting data will provide previously unavailable insights into basic biological processes important for the formation of cells, tissues, organs, and organisms. These research efforts serve as the foundation for interdisciplinary training of diverse participants at the host university, as well as conferences, seminars, and national training centers such as Cold Spring Harbor Laboratory, NY. This work will train a new generation of scientists in both biology and chemistry, including many traditionally underrepresented populations in science, to allow them to address current challenges but also to ask new questions in these fields to better understand normal vertebrate embryo development and diseases.Technical ParagraphDecades of research has uncovered many genes and gene products with critical roles during development of the vertebrate embryo, but how small molecules (called metabolites) participate in cell developmental processes is not fully known. The PI's laboratory recently discovered metabolites capable of altering the normal dorsal-ventral fate of select stem cells in the early frog (Xenopus laevis) embryo, demonstrating that these molecules, too, are active players during patterning of the vertebrate body. The overall goal of this work is to determine the mechanism of action underlying metabolite-induced cell fate decisions. This will be accomplished through a systems cell biology approach, in which the molecular state of metabolite-injected cells will be characterized using unique single-cell mass spectrometry technologies that were developed and validated in the PI's laboratory. The project will identify how cell-fate altering metabolites perturb close-proximity metabolic networks as well as key proteins of metabolism and known signaling pathways of dorsal-ventral specification. Two single-cell mass spectrometry instruments will be used to perform flux analysis for the injected metabolites and to measure the relative translation of targeted proteins in fluorescently tracked cell clones that form from the metabolite-injected cells in the living frog embryo. The resulting data will identify gene candidates for functional tests via gene knock-down experiments to validate the proposed mechanism of action for metabolite-induced cell fate changes. Understanding small-molecule effects on cell fate commitment raises broad implications in diverse areas of the life sciences. The work will also train underrepresented groups in techniques bridging biology and analytical chemistry.